1. Field of the Invention
The present invention relates to a chimeric protein serving as a substrate in the production of a physiologically active peptide or a precursor thereof, and a process for producing the physiologically active peptide or a precursor thereof. Further, the present invention relates to a method for separating the physiologically active peptide from the chimeric protein to purify the physiologically active peptide.
2. Related Art
A number of peptide production processes utilizing the expression of a chimeric protein have been attempted. In this case, chemical or enzymatic cleavage is used for separating a target peptide. Enzymes used in this method include lysyl-endopeptidase (Achromobacter protease I) for specifically cleaving the peptide bond on the C-terminal side of a lysine residue and Staphylococcul protease V8 for specifically cleaving the C-terminal side of a glutamic acid. Chemical methods include cleaving of an asparagine residue by nitrous acid and cleaving of a methionine residue by CNBr.
For the chemical methods, the modification of the target peptide is unavoidable, necessitating the separation of the target peptide from various analogues in the purification of the target peptide after cleaving. On the other hand, for the enzymatic cleaving method, the specificity is high, and the cleaving is performed under relatively moderate conditions, facilitating the purification of the target peptide. Since, however, whether or not enzymes are usable depends upon the amino acid sequence of the target peptide, existing proteases cannot be always used, leading to a demand for universal cleavage enzymes.
When a peptide hormone or a precursor thereof is produced in an organism, a precursor polypeptide for the peptide is specifically cleaved by an enzyme (a processing enzyme). Examples of such enzymes known in the art include a prohormone enzyme 1/3 (PC 1/3), a prohormone enzyme 2 (PC 2), and furin. Kex2 protease which functions in the production of an .alpha.-mating factor of Saccharomyces cerevisiae is also a kind of the above enzyme. When these processing enzymes are used for excising a target peptide from the chimeric protein, it is expected that the peptide hormone is not damaged and the processing enzymes is applicable to a wide variety of peptides. Therefore, the development of such production methods has been desired in the art.
The present inventors have reported a method which comprises providing a polypeptide, derived from E. coli .beta.-galactosidase, as a protective peptide, selecting a proper linker peptide, and efficiently producing a chimeric protein with a target peptide, as an insoluble inclusion body (Japanese Unexamined Patent Publication (Kokai) No. 5-328992). This chimeric protein is solubilized with a modifier, such as urea, and used as a substrate for processing enzymes.